1. Field of Invention
The present invention relates to the microbiological industry, and specifically to a method for producing succinic acid using yeast belonging to the genus Yarrowia in which the activity of succinate dehydrogenase is reduced.
2. Description of the Related Art
Yarrowia lipolytica is a unique yeast due to its ability to produce a wide spectrum of organic acids, including tricarboxylic acid cycle intermediates, such as citric and isocitric acids, and to secrete them into the medium. During continuous cultivation of Y. lipolytica N 1, oxygen requirements for growth and citric acid synthesis were found to depend on the iron concentration in the medium. A coupled effect of oxygen and iron concentrations on the functioning of the mitochondrial electron transport chain in Y lipolytica N 1 was established. Based on the results obtained in continuous culture, conditions for citric acid production in a batch culture of Y. lipolytica N 1 have been proposed (Kamzolova S. V. et al, FEMS Yeast Res.; 3 (2):217-22 (2003)).
Succinic acid, a member of the C4-dicarboxylic acid family, is widely used in the production of foods, pharmaceuticals, and biodegradable plastics. Traditionally, it is produced via chemical synthesis from petrochemical feedstocks that are nonrenewable, and these chemical processes cause environment pollution. Therefore, great attention has been paid to the use of effective natural succinic acid producers, such as microorganisms.
Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where Escherichia coli undergoes mixed-acid fermentation. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently, and the system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network (Lin H. et al., Biotechnol Bioeng; 89(2):148-56 (2005)).
Various E. coli mutant strains designed for succinate production under aerobic conditions were characterized in chemostat. The metabolite profiles, enzyme activities, and gene expression profiles were studied to better understand the metabolic network operating in these mutant strains. The most efficient succinate-producing mutant strain HL27659k has the five following mutations: sdhAB, (ackA-pta), poxB, iclR, and ptsG. It was shown that the succinate synthesis pathways engineered in strain HL27659k were highly efficient, yielding succinate as the only major product produced under aerobic conditions (Lin H. et al., Metab Eng.; 7 (5-6):337-52 (2005)).
Fed-batch reactor experiments were performed for the strain HL27659k(pKK313) under aerobic conditions to determine and demonstrate its capacity for high-level succinate production. Results showed that the aerobic succinate production system using the designed strain HL27659k(pKK313) is more practical than conventional anaerobic succinate production systems. It has a remarkable potential for industrial-scale succinate production and process optimization (Lin H. et al., Biotechnol Bioeng; 90(6):775-9 (2005)).
A two-stage culture of NZN111, pflB ldhA double mutant, which loses its ability to ferment glucose anaerobically due to a redox imbalance, was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while the activities of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. These results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain (Wu H. et al., Appl Environ Microbiol.; 73(24):7837-43 (2007)).
Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. The metabolically engineered LPK7 strain was able to produce succinic acid from glucose with little or no formation of acetic, formic, and lactic acids (Lee S. J. et al., Appl Environ Microbiol.; 72(3):1939-48 (2006)).
Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and the culture pH. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced when grown with excess-CO2—HCO3−, which suggests that there is a threshold level of CO2 for enhanced succinate production in A. succiniciproducens. It was shown that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO2—HCO3− as a consequence of regulating electron sink metabolism (Samuelov N. S. et al., Appl Environ Microbiol.; 57 (10):3013-9 (1991)).
Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing Actinobacillus succinogenes. A. succinogenes growth trends and end product distributions in AM3 and rich medium fermentations were compared. The inability to synthesize alpha-ketoglutarate from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in A. succinogenes (McKinlay J. B. et al., Appl Environ Microbiol.; 71 (11): 6651-6 (2005)).
A method for producing high amounts of succinic acid under anaerobic conditions using E. coli strains, wherein the adhE, ldhA, iclR, arcA, and/or ack-pta genes are disrupted has been disclosed (US 20060046288 A1).
A method of producing succinic acid from industrial-grade hydrolysates by supplying an organism (E. coli, Klebsiela, Erwinia, Lactobacillus) that contains mutations in the genes ptsG, pflB, and ldhA, and allowing the organism to accumulate biomass and metabolize the hydrolysate has been disclosed (US 20030017559 A1).
A fermentation process for producing succinic acid by selecting a bacterial strain that does not produce high yields of succinic acid, disrupting the normal regulation of sugar metabolism of the bacterial strain, and combining the mutant bacterial strain and selected sugar in anaerobic conditions to facilitate production of succinic acid has been described. Also described is a method for changing low-yield succinic acid-producing bacteria to high-yield succinic acid-producing bacteria by selecting a bacterial strain having a phosphotransferase system and altering the phosphotransferase system so as to allow the bacterial strain to simultaneously metabolize different sugars (U.S. Pat. No. 6,159,738).
Succinate dehydrogenase (SDH) of Saccharomyces cerevisiae is composed of four subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. It was determined that double disruption of the SDH1 and SDH2, or SDH1b (the SDH1 homologue) genes is required for complete loss of SDH activity and that the SDH1b gene compensates for the function of the SDH1 gene. The strain with disrupted sdh1 sdh1b genes showed an increase in succinate productivity only up to 1.9-fold along with a decrease in malate productivity relative to the wild-type strains under shaking conditions (Kubo Y. et al., J Biosci Bioeng; 90 (6):619-24 (2000)).
The pathway leading to accumulation of succinate was examined in liquid culture in the presence of a high concentration (15%) of glucose under aerobic and anaerobic conditions using a series of Saccharomyces cerevisiae strains in which various genes that encode the enzymes required in the TCA cycle were disrupted. Results indicate that succinate could be synthesized through two pathways, namely, alpha-ketoglutarate oxidation via the TCA cycle and fumarate reduction under anaerobic conditions (Arikawa Y. et al., J Biosci Bioeng; 87 (1):28-36 (1999)).
Methods for preparing succinic acid using a microorganism transformed with a recombinant vector containing the gene encoding a malic enzyme B (maeB). fumarate hydratase C (fumC), or formate dehydrogenases D & E (fdhD and fdhE) have been disclosed (US 20070042476 A1, US 20070042477 A1, and US 20080020436 A1, respectively).
The set of metabolic modifications responsible for coupling succinate production to the growth of the microorganism include disruption of one or more of the following genes (a) adhE, ldhA; (b) adhE, ldhA, acka-pta; (c) pfl, ldhA; (d) pfl, ldhA, adhE; (e) acka-pta, pykF, atpF, sdhA; (f) acka-pta, pykF, ptsG, or (g) acka-pta, pykF, ptsG, adhE, ldhA. (US 20070111294).
But currently, there have been no reports of reducing the activity of succinate dehydrogenase in a yeast belonging to the genus Yarrowia for the purpose of producing succinic acid.